Maxiprep using alkaline lysis, very high OD, but low intensity on gel - (Aug/06/2010 )
I used alkaline lysis protocol (from molecular cloning) followed by RNase digestion, p:c:i extraction and isopropanol precipitation. Everything looks normal, except during precipitation, the solution became very very dirty and opaque. After air dry, the pellet does become transparent. Nanodrop gives extremely high OD (starting with 500ml culture, low copy plasmid, got ~4000ug/ul in 2ml), with normal OD260/280 (1.8-1.9). However, the most wierd part is when I run the gel to check the quality, the band is defintely not as strong as it should be according to the nanodrop measurement. I compared with my previous Qiagen minipreped plasmid, the gel shows it's only about 200ug/ul (so 1/20 of the nanodrop measurement). I didn't see any other contaminating bands, or RNA from the gel. I am wondering what's happening, what is the things contaminating my plasmid and how should I get rid of it?
I used this method as I heard the efficiency of Qiagen maxiprep kit is low. So I tried the traditional method. But it turns out to be more complicated than I thought...
Thank you so much for any advice!
linyan on Fri Aug 6 17:49:52 2010 said:
I used alkaline lysis protocol (from molecular cloning) followed by RNase digestion, p:c:i extraction and isopropanol precipitation. Everything looks normal, except during precipitation, the solution became very very dirty and opaque. After air dry, the pellet does become transparent. Nanodrop gives extremely high OD (starting with 500ml culture, low copy plasmid, got ~4000ug/ul in 2ml), with normal OD260/280 (1.8-1.9). However, the most wierd part is when I run the gel to check the quality, the band is defintely not as strong as it should be according to the nanodrop measurement. I compared with my previous Qiagen minipreped plasmid, the gel shows it's only about 200ug/ul (so 1/20 of the nanodrop measurement). I didn't see any other contaminating bands, or RNA from the gel. I am wondering what's happening, what is the things contaminating my plasmid and how should I get rid of it?
I used this method as I heard the efficiency of Qiagen maxiprep kit is low. So I tried the traditional method. But it turns out to be more complicated than I thought...
Thank you so much for any advice!
It is likely that your plasmid prep is contaminated by small fragments of DNA, sheared DNA. This type of contamination is made more severe by rough handling of the lysed cell solution solution (after the alkaline solution is added to the E coli which had been resuspended in solution I).
You can cut down this DNA contamination by doing a PEG precipitation.
PEG solution
13% PEG 8000
1.6M NaCl
Resuspend DNA pellet in 400ul.
Add 400ul PEG solution
Keep in freezer for 15min
Spin down at maximum rpm for 12min.
(There should be a small almost translucent pellet at the bottom of the tube.)
Wash pellet in 70% ethanol.
Then resuspend in TE.
It happened to me when I was new in Gigaprep. I think this is genomic DNA contamination since the genomic has high MW so it is difficult to dissolve. Remember that you cannot apply any UV measurment for plasmid until you get a clear solution because the instrument will detect the cloud as absorbance so it will give you high OD. to estimate the real concentration you may centrifuge 13000 G for 10-15 min, then take the supernatant and measure the OD.